The present invention relates to cycloalkano-indole and -azaindole derivatives, processes for their preparation and their use as medicaments, in particular as antiatherosclerotic medicarnents.
It is known that increased blood levels of triglycerides (hypertriglyceridaemia) and cholesterol (hypercholesterolaemia) are associated with the genesis of atherosclerotic vessel wall changes and coronary heart diseases.
A distinctly increased risk of the development of coronary heart disease is moreover present if these two risk factors occur in combination, which is accompanied, in turn, with an overproduction of apolipoprotein B-100. There is therefore, as before, a great need to make available effective medicarnents for the control of atherosclerosis and coronary heart diseases.
The present invention relates to cycloalkano-indole and -azaindole derivatives of the general formula (I) 
in which
R1 and R2, including the double bond connecting them, together form a phenyl or pyridyl ring or a ring of the formula 
wherein
R8 denotes hydrogen or straight-chain or branched alkyl having up to 4 carbon atoms,
R3 and R4, including the double bond connecting them, together form a phenyl ring or a 4- to 8-membered cycloaukene or oxocycloalkene radical,
all ring systems mentioned under R1/R2 and R3/R4 optionally being substituted up to 3 times by identical or different halogen, trifluoromethyl, carboxyl or hydroxyl substituents, by straight-chain or branched alkoxy or alkoxycarbonyl each having up to 6 carbon atoms or by straiht-chain or branched alkyl having up to 6 carbon atoms, which, for its part, can be substituted by hydroxyl or by straight-chain or branched alkoxy having up to 4 carbon atoms,
D represents hydrogen, cycloalkyl having 4 to 12 carbon atoms or straight-chain or branched alkyl having up to 12 carbon atoms,
E represents the xe2x80x94COxe2x80x94 or xe2x80x94CSxe2x80x94 group,
L represents an oxygen or sulphur atom or a group of the formula xe2x80x94NR9,
wherein
R9 denotes hydrogen or straight-chain or branched alkyl having up to 6 catbon atoms, which is optionally substituted by hydroxyl or phenyl,
R5 represents phenyl or a 5- to 7-membered sated or unsaturated heterocycle having up to 3 heteroatoms from the series consisting of S, N and/or O, the cycles optionally being substituted up to 3 times by identical or different nitro, carboxyl, halogen or cyano substituents or by straight-chain or branched alkenyl or alkoxycarbonyl each having up to 6 carbon atoms or by staight-chain or branched alkyl having up to 6 carbon atoms, which is optionally substituted by hydroxyl, carboxyl or by straight-chain or branched alkoxy or alkoxycarbonyl each having up to 6 carbon atoms, and/or the cycles optionally being substituted by a group of the formula xe2x80x94OR10 or xe2x80x94NR11R12,
wherein
R10 denotes hydrogen or straight-chain or branched alkyl or alkenyl each having up to 6 carbon atoms,
R11 and R12 are identical or different and denote phenyl, hydrogen or straight-chain or branched alkyl having up to 6 carbon atoms or straight-chain or branched acyl having up to 8 carbon atoms, which is optionally substituted by a group of the formula xe2x80x94NR13R14,
wherein
R13 and R14 are identical or different and denote hydrogen or straight-chain or branched acyl having up to 8 carbon atoms,
R6 represents hydrogen, carboxyl or straight-chain or branched alkoxycarbonyl having up to 5 carbon atoms, or represents straight-chain or branched alkyl having up to 6 carbon atoms, which is optionally substituted by hydroxyl or by a group of the formula xe2x80x94Oxe2x80x94COxe2x80x94R15,
wherein
R15 denotes phenyl which is optionally substituted up to 3 times by identical or different halogen or hydroxyl substituents or by straight-chain or branched alkyl having up to 5 carbon atoms, or straight-chain or branched alkyl or alkenyl each having up to 22 carbon atoms, each of which is optionally substituted by a group of the formula xe2x80x94OR6,
wherein
R16 is hydrogen, benzyl, triphenylmethyl or straight-chain or branched acyl having up to 6 carbon atoms,
R7 represents hydrogen or
R6 and R7 together represent the group of the formula xe2x95x90O,
if appropriate in an isomeric form and their salts.
The cycloalkan-indole and -azaindole derivatives according to the invention can also be present in the form of their salts. In general, salts with organic or inorganic bases or acids may be mentioned here.
In the context of the present invention, physiologically acceptable salts are preferred. Physiologically acceptable salts of the compounds according to the invention can be salts of the substances according to the invention with mineral acids, carboxylic acids or sulphonic acids. Particularly preferred salts are, for example, those with hydrochloric acid, hydrobrornic acid, sulphuric acid, phosphoric acid, methanesulphonic acid, ethanesulphonic acid, toluenesulphonic acid, benzenesulphonic acid, naphthalenedisulphonic acid, acetic acid, propionic acid, lactic acid, tartaric acid, citric acid, fumaric acid, maleic acid or benzoic acid.
Physiologically acceptable salts can also be metal or ammonium salts of the compounds according to the invention which have a free carboxyl group. Particularly preferred salts are, for example, sodium, potassium, magnesium or calcium salts, and also ammonium salts which are derived from ammonia, or organic amines, such as, for example ethylamine, di- or triethylamine, di- or triethanolamine, dicyclohexylamine, dimethylaminoethanol, arginine, lysine, ethylenediamine or 2-phenylethylamine.
Including the double bond of parent structure, the cycloalkene radical (R3/R4) in the context of the invention in general represents a 4 -to 8-membered hydrocarbon radical, preferably a 5- to 8-membered hydrocarbon radical, for example a cyclobutene, cyclopentene, cyclohexene, cycloheptene or cyclooctene radical. The cyclopentene, cyclohexene, cyclooctene or cycloheptene radicals are preferred
Heterocycle (R5) in the context of the invention in general represents a saturated or unsaturated 5- to 7-membered heterocycle, preferably a 5- to 6-membered heterocycle, which can contain up to 3 heteroatoms from the series consisting of S, N and/or O. Examples which may be mentioned are: pyridyl, thienyl, furyl, pyrrolyl, thiazolyl, oxazolyl, imidazolyl, morpholinyl or piperidyl. Pyridyl and thienyl are preferred.
The compounds according to the invention can exist in stereoisomeric forms which either behave as image and mirror image (enantiomers), or do which do not behave as image and mirror image (diastereomers). The invention relates both to the enantiomers and diastereomers and their respective mixtures. These mixtures of the enantiomers and diastereomers can be separated in a known manner into the stereoisomerically uniform constituents.
Preferred compounds of the general formula (I) are those
in which
R1 and R2, including the double bond connecting them, together form a phenyl or pyridyl ring or a ring of the formula 
wherein
R8 denotes hydrogen or stright-chain or branched alkyl having up to 3 carbon atoms,
R3 and R4, including the double bond connecting them, together form a phenyl ring or a cyclopentene, cyclohexene, cycloheptene, cyclooctene, oxocyclopentene, oxocyclohexene, oxocycloheptene or oxocyclooctene radical,
all ring systems mentioned under R1/R2 and R3/R4 optionally being substituted up to 2 times by identical or different fluorine, chlorine, bromine, trifluoromethyl, carboxyl or hydroxyl substituents, by straight-chain or branched alkoxy or alkoxycarbonyl each having up to 4 carbon atoms or by straight-chain or branched alkyl having up to 4 carbon atoms, which, in turn, can be substituted by hydroxl or by straight-chain or branched alkoxy having up to 3 carbon atoms.
D represents hydrogen, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl or straight-chain or branched alkyl having up to 10 carbon atoms,
E represents the xe2x80x94COxe2x80x94 or xe2x80x94CSxe2x80x94 group,
L represents an oxygen or sulphur atom or represents a group of the formula xe2x80x94NR9,
wherein
R9 denotes hydrogen or staight-chain or branched alkyl having up to 5 carbon atoms, which is optionally substituted by hydroxyl or phenyl,
R5 represents phenyl, pyridyl, furyl, thienyl or imidazolyl, each of which is optionally substituted up to 2 times by identical or different nitro, carboxyl, fluorine, chlorine, bromine or cyano substituents, by straight-chain or branched alkenyl or alkoxy carbonyl each having up to 4 carbon atoms or by straight-chain or branched alkyl having up to 5 carbon atoms, which is optionally substituted by hydroxyl, carboxyl or by staight-chain or branched alkoxy or alkoxycarbonyl each having up to 5 carbon atoms, and/or the cycles are optionally substituted by a group of the formula xe2x80x94OR10 or xe2x80x94NR11R12,
wherein
R10 denotes hydrogen or straight-chain or branched alkyl or alkenyl each having up to 4 carbon atoms,
R11 and R12 are identical or different and denote phenyl, hydrogen or straight-chain or branched alkyl having up to 5 carbon atoms or denote straight-chain or branched acyl having up to 6 carbon atoms, which is optionally substituted by a group of the formula xe2x80x94NR13R14,
wherein
R13 and R14 are identical or different and denote hydrogen or straight-chain or branched acyl having up to 6 carbon atoms,
R6 represents hydrogen carboxyl or straight-chain or branched alkoxycarbonyl having up to 4 carbon atoms, or represents straight-chain or branched alkyl having up to 5 carbon atoms, which is optionally substituted by hydroxyl or by a group of the formula xe2x80x94Oxe2x80x94COxe2x80x94R15,
wherein
R15 denotes phenyl which is optionally substituted up to 3 times by identical or different fluorine, chlorine, bromine or hydroxyl substituents or by straight-chain or branched alkyl having up to 4 carbon atoms, or straight-chain or branched alkyl or alkenyl each having up to 20 carbon atoms, each of which is optionally substituted by a group of the formula xe2x80x94OR16,
wherein
R16 is hydrogen, benzyl, triphenylmethyl or straight-chain or branched acyl having up to 5 carbon atoms,
R7 represents hydrogen or
R6 and R7 together represent the group of the formula xe2x95x90O,
if appropriate in an isomeric form, and their salts.
Particularly preferred compounds of the general formula (I) are those
in which
R1 and R2, including the double bond connecting them, together form a phenyl or pyridyl ring or a ring of the formula 
wherein
R8 denotes hydrogen or methyl,
R3 and R4, including the double bond connecting them, together form a phenyl ring or a cyclopentene, cyclohexene, cycloheptene, cyclooctene, oxocyclopentene, oxocyclohexene, oxocycloheptene or oxocyclooctene radical,
all ring systems mentioned under R1/R2 and R3/R4 optionally being substituted up to 2 times by identical or different fluorine, chlorine, bromine, trifluoromethyl, carboxyl or hydroxyl substituents, by straight-chain or branched alkoxy or alkoxycarbonyl each having up to 3 carbon atoms or by straight-chain or branched alkyl having up to 3 carbon atoms, which, for its part, can be substituted by hydroxyl, methoxy or ethoxy,
D represents hydrogen, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl or straight-chain or branched alkyl having up to 6 carbon atoms,
E represents the xe2x80x94COxe2x80x94 or xe2x80x94CSxe2x80x94 group,
L represents an oxygen or sulphur atom or represents a group of the formula xe2x80x94NR9,
wherein
R9 denotes hydrogen or straight-chain or branched alkyl having up to 4 carbon atoms, which is optionally substituted by hydroxyl or phenyl,
R5 represents phenyl, pyridyl or thienyl, each of which is optionally substituted up to 2 times by identical or different nitro, carboxyl, fluorine, chlorine, bromine or cyano substituents, by straight-chain or branched alkenyl or alkoxycarbonyl each having up to 3 carbon atoms or by straight-chain or branched alkyl having up to 4 carbon atoms, which is optionally substituted by hydroxyl, carboxyl or by straight-chain or branched alkoxy or alkoxycarbonyl each having up to 4 carbon atoms, and/or the cycles are optionally substituted by a group of the formula xe2x80x94OR10 or xe2x80x94NR11R12,
wherein
R10 denotes hydrogen or straight-chain or branched alkyl or alkenyl each having up to 3 carbon atoms,
R11 and R12 are identical or different and denote phenyl, hydrogen or straight-chain or branched alkyl having up to 4 carbon atoms or denote straight-chain or branched acyl having up to 5 carbon atoms, which is optionally substituted by a group of the formula xe2x80x94NR13R14,
wherein
R13 and R14 are identical or different and denote hydrogen or straight-chain or branched acyl having up to 5 carbon atoms,
R6 represents hydrogen, carboxyl or straight-chain or branched alkoxycarbonyl having up to 3 carbon atoms, or represents straight-chain or branched alkyl having up to 4 carbon atoms, which is optionally substituted. by hydroxyl or by a group of the formula xe2x80x94Oxe2x80x94COxe2x80x94R15,
wherein
R15 denotes phenyl which is optionally substituted up to 3 times by identical or different straight-chain or branched alkyl having up to 3 carbon atoms, or denotes straight-chain or branched alkyl or alkenyl each having up to 19 carbon atoms, each of which is optionally substituted by a group of the formula xe2x80x94OR16,
wherein
R16 denotes hydrogen, benzyl, triphenylmethyl or straight-chain or branched acyl having up to 4 carbon atoms,
R7 represents hydrogen or
R6 and R7 together represent the group of the formula xe2x95x90O,
if appropriate in an isomeric form, and their salts.
A process for the preparation of the compounds of the general formula (I) according to the invention has additionally been found, characterized in that carboylic acids of the general formula (II) 
in which
R1, R2, R3, R4 and D have the meaning indicated, are amidated using compounds of the general formula (III) 
in which
R5 has the meaning indicated
and
R17 has the indicated meanig of R6, but does not represent carboxyl,
in an inert solvent and in the presence of bases and/or auxiliaries,
and, if appropriate, functional groups are varied by hydrolysis, esterification or reduction.
The process according to the invention can be illustrated by the following reaction scheme: 
Suitable solvents for the amidation are in this case inert organic solvents which do not change under the reaction conditions. These include ethers, such as diethyl ether or tetrahydrofuran halogenohydrcarbons such as dichloromethane, trichioromethane, tetrachloromethane, trichloroethane, tetrachloroethane, 1 ,2-dichloroethane or trichloroethylene, hydrocarbons such as benzene, xylene, toluene, hexane, cyclohexane or petroleum fractions, nitromethane, dimethylformamide, acetone, acetonitrile or hexamethylphosphoramide. It is also possible to employ mixtures of the solvents. Dichloromethane, tetrahydrofuran, acetone and dimethylformamide are particularly preferred.
Bases which can be employed for the process according to the invention are in general inorganic or organic bases. These preferably include alkali metal hydroxides such as, for example, sodium hydroxide or potassium hydroxide, alkaline earth metal hydroxides, such as, for examnple, barium hydroxide, alkali metal carbonates such as sodium carbonate or potassium carbonate, alkaline earth metal carbonates such as calcium carbonate, or alkali metal alkoxides such as sodium or potassium methoxide, sodium or potassium ethoxide or potassium tert-butoxide, or organic amines (trialkyl(C1-C6)amines) such as triethylamine, or heterocycles such as 1,4diazabicyclo[2.2.2]octane (DABCO), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), pyridine, diaminopyridine, methylpiperidine or morpholine. It is also possible to employ alkali metals such as sodium and their hydrides such as sodium hydride as bases. Sodium and potassium carbonate and triethylamine are preferred.
The base is employed in an amount from 1 mol to 5 mol, preferably from 1 mol to 3 mol, relative to 1 mol of the compound of the general formula (II).
The reaction is in general carried out in a temperature range from 0xc2x0 C. to 150xc2x0 C., preferably from +20xc2x0 C. to +110xc2x0 C.
The reaction can be carried out at normal, increased or reduced pressure (e.g. 0.5 to 5 bar). In general, the reaction is carried out at normal pressure.
The amidation can optionally proceed via the activated stage of the acid halides, which can be prepared from the corresponding acids by reaction with thionyl chloride, phosphorus trichloride. phosphorus pentachloride, phosphorus tribromide or oxalyl chloride.
The abovementioned bases can optionally also be employed for the amidation as acid-binding auxiliaries.
Suitable auxiliaries are also dehydrating reagents. These include, for exarnple, carbodiimides such as diisopropylcarbodiimide, dicyclohexylcarbodiimide and N-(3-dimethylaminopropyl)Nxe2x80x2-ethylcarbodiimide hydrochloride or carbonyl compounds such as carbonyldiidazole or 1,2-oxazolium compounds such as 2-ethyl-5-phenyl-1 ,2-oxazolium-3-sulphonate or propanephosphonic anhydride or isobutyl chloroformate or benzotriazolyloxy-tris-dimethylamino)phosphonium hexafluorophosphate or diphenyl phosphoramidate or methanesulphonyl chloride, if appropriate in the presence of bases such as triethylamine or N-ethylmorpholine or N-methylpiperidine or dicyclohexylcarbodiimide and N-hydroxysuccinimide.
The acid-binding agents and dehydrating reagents are in general employed in an amount from 0.5 to 3 mol, preferably from 1 to 1.5 mol, relative to 1 mol of the corresponding carboxylic acids.
The variation of functional groups, for example hydrolysis, esterification and reduction, and also separation of isomers and salt formation is carried out by customary methods.
The carboxylic acids of the general formula (II) are new and can be prepared by reacting compounds of the general formula (IV) 
in which
D has the meaning indicated,
T represents a typical leaving group, for example chlorine, bromine, iodine, tosylate or mesylate, preferably bromine,
and
R18 represents (C1-C4)-alkyl, with compounds of the general formula (V) 
in which
R1, R2, R3 and R4 have the meaning indicated, in inert solvents, if appropriate in the presence of a base.
Suitable solvents for the process are the customary organic solvents which do change under the reaction conditions. These preferably include ethers such as diethyl ether, dioxane, tetrahydrofuran, glycol dimethyl ether, or hydrocarbons such as benzene, toluene, xylene, hexane, cyclohexane or petroleum fractions, or halogenohydrocarbons such as dichloromethane, trichloromethane, tetrachloromethane, dichloroethylene, trichloroethylene or chlorobenzene, or ethyl acetate, triethylamine, pyridine, dimethyl sulphoxide, dimethylformamide, hexamethylphosphoramide, acetonitrile, acetone or nitromethane. It is also possible to use mixtures of the solvents mentioned. Dimethylformamide and tetrahydrofuran are preferred.
The bases employed for the process according to the invention can in general be inorganic or organic bases. These preferably include alkali metal hydroxides, for example, sodium hydroxide or potassium hydroxide, alkaline earth metal hydroxides, for example, barium hydroxide, alkali metal carbonates such as sodium carbonate or potassium carbonate, alkaline earth metal carbonates such as calcium carbonate, or alkali metal alkoxides such as sodium or potassium methoxide, sodium or potassium ethoxide or potassium tert-butoxide, or organic amines (trialkyl(C1-C6)amines) such as triethylamine, or heterocycles such as 1,4diazabicyclo[2.2.2]octane (DABCO), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), pyridine, diamninopyridine, methylpiperidine or morpholine. It is also possible to employ alkali metals such as sodium or their hydrides such as sodium hydride as bases. Sodium hydride, potassium carbonate, triethylamine, pyridine and potassium tert-butoxide, DBU or DABCO are preferred.
In general, the base is employed in an amount from 0.05 mol to 10 mol, preferably from 1 mol to 2 mol, relative to 1 mol of the compound of the formula (IV).
The process according to the invention is in general carried out in a temperature range from xe2x88x9230xc2x0 C. to +100xc2x0 C., preferably from xe2x88x9210xc2x0 C. to +60xc2x0 C.
The process according to the invention is in general carried out at normal pressure. However, it is also possible to carry out the process at elevated pressure or at reduced pressure (e.g. in a range from 0.5 to 5 bar).
The compounds of the general formula (III) are known per se.
The compounds of the general formula (IV) are known or can be prepared in analogy to known methods.
The compounds of the general formula (V) are known or can be prepared in analogy to known methods.
The compounds of the general formula (I) according to the invention have an unforeseeable spectrum of pharmacological action.
They can be used as active compounds in medicaments for the reduction of changes to vessel walls and for the treatment of coronary heart disorders, cardiac insufficiency, brain power disorders, ischaernic brain disorders, apoplexy, circulatory disorders, disorders of the microcirculation and thromboses.
Furthermore, the proliferation of smooth muscle cells plays a decisive part in the occlusion of vessels. The compounds according to the invention are suitable for inhibiting this proliferation and thus preventing atherosclerotic processes.
The compounds according to the invention are distinguished by a lowering of the ApoB-100-associated lipoproteins (VLDL and its degradation products, e.g. LDL), of ApoB-100, of triglycerides and of cholesterol. They thus have useful, superior pharmacological properties in comparison with the prior art.
Surprisingly, the action of the compounds according to the invention consists first in a decrease or complete inhibition of the formation and/or the release of ApoB-100-associated lipoproteins from liver cells, which results in a lowering of the VLDL plasma level. This lowering of VLDL must be accompanied by a lowering of the plasma level of ApoB100, LDL, triglycerides and cholesterol; a number of the abovementioned risk factors which are involved in vessel wall changes are thus simultaneously decreased.
The compounds according to the invention can therefore be employed for the prevention and treatment of atherosclerosis, obesity, pancreatitis and constipation.
I Inhibition of the release of ApoB-100 Associated Lipopnteins
The test for detecting the inhibition of the release of ApoB-100-associated lipoproteins from liver cells was carried out in vitro using cultured liver cells, preferably using cells of the human line HepG2. These cells are cultured under standard conditions in medium for the culture of eukaryotic cells, preferably in RPMI 1640 With 10% foetal calf serum. HepG2 cells synthesize and secrete into the culture supernatant ApoB-100-associated lipoprotein particles which in principle are built up in a similar manner to the VLDL and LDL particles which are to be found in the plasma
These particles can be detected using an immunoassay for human LDL. This immunoassay is carried out using antibodies which have been induced against human LDL in rabbits under standard conditions. The anti-LDL antibodies (rabbit anti-LDL Ab) wevere purified by affiity chromatography on an inununosorbent using human LDL. These purified rabbit anti-LDL Ab are adsorbed on the surface of plastic. Expediently, this adsorption is carried out on the plastic surface of microtitre plates having 96 wells, preferably on MaxiSorp plates. If ApoB-100-associated particles are present in the supernatant of HeG2 cells, they can be bound to the insolubilized rabbit anti-LDL Ab, and an immune complex results which is bound to the plastic surface. Unbound proteins are removed by washing. The immune complex located on the plastic surface is detected using monoclonal antibodies which have been induced against human LDL and purified according to standard conditions. These antibodies were conjugated with the enzyme peroxidase. Peroxidase converts the colourless substrate TMB into a coloured product in the presence of H2O2. After acidification of the reaction mixture with H2SO4, the specific light absorption at 450 nm is detemined, which is a measure of the amount of ApoB-100associated particles which have been secreted into the culture supernatant by the HepG2 cells.
Surprisingly, the compounds according to the invention inhibit the release of the ApoR100-associated particles. The IC50 value indicates at which substance concentration the light absorption is inhibited by 50% in comparison with the control (solvent control without substance).
2. Determination of the VLDL Secretion in vivo in the Hamster
The effect of the test substances on VLDL secretion in vivo is investigated in the hamster. To do this, golden hamsters are anaesthetized with Ketaset (83 mg/kg s.c.) and Nembutal (50 mg/kg i.p.) after premedication with atropine (83 mg/kg s.c.). When the animals have become reflex-free, the jugular vein is exposed and cannulated. 0.25 ml/kg of a 20% strength solution of Triton WR-1339 in physiological saline solution is then administered. This detergent inhibits the lipoprotein lipase and thus leads to a rise in the triglyceride level as a result of a lack of catabolism of secreted VLDL particles. This triglyceride rise can be used as a measure of the VLDL secretion rate.
Blood is taken from the animals before and also one and two hours after administration of the detergent by puncture of the retroorbital venous plexus. The blood is incubated for two hours at room temperature, and then overnight at 4xc2x0 C., in order to end clotting completely. It is then centrifuged at 10,000 g for 5 minutes. The triglyceride concentration in the serum thus obtained is determined with the aid of a modified commercially available enzyme test (Merckotest(copyright) triglyceride No. 14354). 100 xcexcl of serum are treated with 100 xcexcl of test reagent in 9whole plates and incubated at room temperature for 10 minutes. The optical density is then determined at a wavelength of 492 nM in an automatic plate-reading apparatus (SLT Spectra). Serum samples having an excessively high triglyceride concentration are diluted with physiological saline solution. The triglyceride concentration contained in the samples is determined with the aid of a standard curve measured in parallel. In this model, test substances are administered intravenously either immediately before administration of the detergent or orally or subcutaneously before initiation of anaesthesia.
3. Inhibition of Intestinal Triglyceride Absorption in vivo (Rats)
The substances which are to be investigated for their triglyceride absorption-inhibiting action in vivo are administered orally to male Wistar rats having a body weight of between 170 and 230 g. For this purpose, the animals are divided into groups of 6 animals 18 hours before substance administration and food is then withdrawn from them. Drking water is available to the animals ad libitum The animals of the control groups receive an aqueous tragacanth suspension or a tragacanth suspension which contains olive oil. The tragacanth-olive oil suspension is prepared using an Ultra-Turrax. The substances to be investigated are suspended in an appropriate tragacanth-olive oil suspension likewise using the Ultra-Turrax, directly before substance administration.
To determine the basal serum triglyceride content, blood is taken from each rat by puncture of the retroorbital venous plexus before stomach tube application. The tragacanth suspension, the tragacanth-olive oil suspensions without substance (control arimals) or the substances suspended in an appropriate tragacanth-olive oil suspension are then administered to the fasting animals using a stomach tube. Further taking of blood to determine the postprandial serum triglyceride rise is carried out, as a rule, 1, 2 and 3 hours after stomach tube application.
The blood samples are centrifuged and, after recovering the serum, the triglycerides are determined photometrically using an EPOS analyzer 5060 (Eppendorf Geratebau, Netheler and Hinz GmbH, Hamburg). The determination of the triglycerides is carried out completely enzymatically using a standard commercial UV test.
The postprandial serum triglyceride rise is determined by subtraction of the triglyceride preliminary value of each animals from its corresponding postprandial triglyceride concentrations (1, 2 and 3 hours after administration).
The differences (in mmouL/) at each point in time (1, 2 and 3 hours) are averaged in the groups, and the mean values of the serum triglyceride rise (xcex94 TG) of the substance-treated animals is compared with the animals which only received the tragacanth-oil suspension.
The serum triglyceride course of the control animals which only received tragacanth is also calculated. The substance effect at each point in time (1, 2 and 3 hours) is determined as follows and indicated in xcex94% of the oil-loaded control.       Δ    ⁢    %    ⁢          xe2x80x83        ⁢    Triglyceride    ⁢          xe2x80x83        ⁢    rise    =                              Δ          ⁢                      xe2x80x83                    ⁢                      TG            substance                          -                  Δ          ⁢                      xe2x80x83                    ⁢                      TG                          tragacanth              ⁢                              xe2x80x83                            ⁢              control                                                            Δ          ⁢                      xe2x80x83                    ⁢                      TG                          oil              ⁢                              xe2x80x83                            ⁢              loading                                      -                  Δ          ⁢                      xe2x80x83                    ⁢                      TG                          tragacanth              ⁢                              xe2x80x83                            ⁢              control                                            xc3x97    100  
Effect of 10 mg of test substance/kg of body weight p.o. on the triglyceride rise (xcex94%) 2 h after a triglyceride loading in the serum of fasting rats. Tlhe serum triglyceride rise of fat-loaded control animals relative to the serum triglyceride level of tragacanth control animals corresponds to 100%. n=6 animals per group.
Statistical evaluation is carried out using Student""s t test after preliminary checking of the variances for homogeneity.
Substances which at one point in time statistically significantly (p less than 0.05) decrease the postprandial serun triglyceride rise by at least 30% compared with the untreated control group are regarded as pharmacologically active.
4. Inhibition of VLDL Secretion in vivo (Rats)
The action of the test substances on VLDL secretion is likewise investigated in the rat. To do this, 500 mg/kg of body weight (2.5 mgkg) of Triton WR-1339, dissolved in physiological saline solution, is administered intravenously into the tail vein of rats. Triton WR-1339 inhibits lipoprotein lipase and thus leads to an increase in the triglyceride and cholesterol level by inhibition of the VLDL catabolism. These rises can be used as a measure of the VLDL secretion rate.
Blood is taken from the animals by puncture of the retroorbital venous plexus before and also one and twvo hours after administration of the detergent. The blood is incubated at room temperature for 1 h for clotting and the serum is obtained by centrifugation at 10,000 g for 20 s. The triglycerides are then photometrically determined by means of a standard commercial coupled enzyme test (Sigma Diagnostics(copyright), No. 339) at a wavelength of 540 nm Measurement is carried out with the aid of a likewise coupled enzyme test (Boehring Mannheim(copyright), No. 1442350) at a wavelength of 546 nm. Samples with triglyceride or cholesterol concentrations which exceed the measuring range of the methods are diluted with physiological saline solution. The determination of the respective serum concentrations is carried out with the aid of standard series measured in parallel. Test substances are administered orally, intravenously or subcutaneously immediately after the Triton injection.
The invention additionally relates to the combination of cycloalkano-indole and -azaindole derivatives of the general formula (1) with glucosidase and/or amylase ihibitor for the treatment of familial hyperlipidaemia, obesity (adiposity) and diabetes mellitus. Glucosidase and/or amylase inhibitors in the context of the invention are, for example, acarbose, adiposine, voglibase, miglitol, emiglitate, MDL 25637, camialibase (MDL 73945), tendamistat, AI-3688, trestatin, pradimnilin-Q and salbostatin.
Combination of acarbose, miglitol, emiglitate or voglibase with one of the abovementioned compounds of the general formula (I) according to the invention is preferred.
The new active compounds can be converted in a known manner into the customary formulations, such as tablets, coated tablets, pills, granules, aerosols, syrups, emulsions, suspensions and solutions, using inert, non-toxic, pharmaceutically suitable excipients or solvents. In this case, the therapeutically active compound should in each case be present in a concentration of approximately 0.5 to 90% by weight of the total mixture, i.e. in amounts which are sufficient in order to achieve the dosage range indicated.
The formulations are prepared, for example, by extending the active compounds with solvents and/or excipients, if appropriate using emulsifiers and/or dispersants, it optionally being possible, e.g. in the case of the use of water as a diluent, to use organic solvents as auxiliary solvents.
Administration is carried out in a customary manner, preferably orally or parenterally, in particular perlingually or intravenously.
In the case of parenteral administration, solutions of the active compound can be employed using suitable liquid vehicles.
In general, it has proved advantageous in the case of intravenous adminstration to administer amounts of approxinately 0.001 to 1 mg/k of body weight, preferably approxijmaely 0.01 to 0.5 mg/kg of body weight, to achieve effective results, and in the case of oral adrministration the dose is approximately 0.01 to 20 mg/kg of body weight, preferably 0.1 to 10 mg/kg of body weight.
In spite of this, it may optionally be necessary to depart from the amounts mentioned, namely depending on the body weight or on the type of administration route, on individual behaviour towards the medicament, the manner of its formulation and the time or interval at which administration takes place. Thus, in some cases it may be adequate to manage with less than the abovementioned minimum amount. while in other cases the upper limit mentioned must be exceeded. In the case of the administration of larger amounts, it may be advisable to divide these into several individual doses over the course of the day.
Definition of the Isomer Types:
4 dia=mixture of the four possible diastereomers in the case of two centres of asymmetry in the molecule
dia A=diastereomer having the larger Rf value
dia B=diastereomer having the smaller Rf value
ent=enantiomer
2 ent dia=mixture of two enantiomerically pure diastereomers
ent dia A=enantiomerically pure diastereomer having the larger Rf value
ent dia B=enantiomerically pure diastereomer having the smaller Rf value
R=R enantiomer
rac=racernate
rac dia A=racemic diastereomer having the larger Rf value
rac dia B=racermic diastereomer having the smaller Rf value
S=S enantiomer
Abbevations Used:
Ac=acetyl
Bn=benzyl
Bz=benzoyl
iB=isobutyl
nBu=normnal butyl
sBu=secondary butyl
tBu=tertiary butyl
DDQ=2,3-dichloro-5,6dicyano-1,4benzoquinone
cDec=cyclodecyl
DMF=N,N-dimethylformamide
DMSO=dimethyl sulphoxide
cDodec=cyclododecyl
Et=ethyl
cHept=cyclo-heptyl
cHex=cyclo-hexyl
HOBT=1-hydroxy-1H-benzotriazole
Me=metlyl
Mes=mesyl
cNon=cyclo-nonyl
cOct=cyclo-octyl
cPent=cyclo-pentyl
nPent=normal pentyl
Ph=phenyl
cPr=cyclo-propyl
nPr=normal propyl
iPr=isopropyl
THF=tetrahydrofuran
TMS=tetramethylsilane
pTol=para-tolyl
pTos=para-tosyl
cUndec=cyclo-undecyl
Solvent Symbol
Dichloromethane: methanol=20:1 A
Dichloromehane: methanol=50:1 B
Dichloromethane: ethanol=20:1 C
Dichiorometiane: ethanol=50:1 D
Petroleum ether: ethyl acetate=1:1 E
Dichloromethane: methanol: acetic acid=90:10:2 F
Petroleum ether: ethyl acetate=2:1 G
Petroleum ether: ethyl acetate=10: 1 H
Toluene I
Toluene: ethyl acetate=1:1 K
Petroleum ether: ethyl acetate=5:1 L
Dichloromethane M
Petroleum ether: ethyl acetate=20:1 N
Dichloromethane: methanol 10:1 0
Cyclohexane: ethyl acetate=1: 1 p
Toluene: ethyl acetate=9:1 Q
Toluene: ethyl acetate=8:1 R
Petroleum ether: ethyl acetate=1:2 S
Dichloromethane: ethanol=5:1 T
Dichloromethane: ethanol=10:1 U
Preparion Procedure for the TLC Mobile Phase BABA:
87.9 ml of an aqueous 0.06667 molar potassium dihydrogen phosphate solution and 12.1 ml of an aqueous 0.06667 molar disodium hydrogen phosphate solution are mixed 60 ml of the solution prepared in this way are shaken with 200 ml of n-butyl acetate, 36 ml of n-butarol and 100 ml of glacial acetic acid and the aqueous phase is removed. The organic phase is the mobile phase BABA.